Interleukin-12 (IL-12) is a heterodimeric cytokine (p70) composed of two subunits (p35 and p40), and plays key roles in immune responses by bridging innate resistance and antigen-specific adaptive immunity. Trinchieri (1993) Immunol Today 14: 335. For example, it promotes type 1 T helper cell (Th1) responses and, hence, cell-mediated immunity. Chan et al. (1991) J Exp Med 173: 869; Seder et al. (1993) Proc Natl Acad Sci USA 90: 10188; Manetti et al. (1993) J Exp Med 177: 1199; and Hsieh et al. (1993) Science 260: 547. Overproduction of IL-12 causes excessive Th1 responses, and may result in inflammatory disorders, insulin-dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, psoriasis, Crohn""s disease, or sepsis. See, for example, Gately et al. (1998) Annu Rev Immunol. 16: 495; and Abbas et al. (1996) Nature 383: 787. Thus, inhibiting IL-12 overproduction is an approach to treat the just-mentioned diseases. Trembleau et al. (1995) Immmunol. Today 16: 383; and Adorini et al. (1997) Chem. Immunol. 68: 175. For example, overproduction of IL-12 and the resultant excessive Th1 type responses can be suppressed by modulating IL-12 production. A compound that down-regulates IL-12 production can be used for treating inflammatory diseases. Ma et al. (1998) Eur Cytokine Netw 9: 54.
This invention is based on the identification of new compounds from a library of 80,000 compounds, which were screened for their abilities to inhibit IL-12 overproduction. In one aspect, this invention features triazine compounds of formula (I) 
wherein R1 is 
[referred to hereinafter as NC(RaRb)], aryl, or heteroaryl; each of R2, R4, and R5, independently, is Rc, halogen, nitro, nitroso, cyano, azide, isothionitro, SRc, or ORc; R3 is Rc, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl, ORc, OC(O)Rc, SO2Rc, S(O)Rc, S(O2)NRcRd, SRc, NRcRd, NRcCORd, NRcC(O)ORd, NRcC(O)NRcRd, NRcSO2Rd, CORc, C(O)ORc, or C(O)NRcRd; n is 0, 1, 2, 3, 4, 5, 6, or 7; X is O, S, S(O), S(O2), or NRc; Y is a covalent bond, CH2, C(O), Cxe2x95x90Nxe2x80x94Rc, Cxe2x95x90Nxe2x80x94ORc, Cxe2x95x90Nxe2x80x94SRc, O, S, S(O), or S(O2); Z is N; and W is O, S, S(O), S(O2), NRc, or NC(O)Rc; in which each of Raand Rb, independently, is H, alkyl, aryl, heteroaryl; and each of Rcand Rd, independently, is H, alkyl, or alkylcarbonyl. Note that the left atom shown in any substituted group described above is closest to the tirazine ring. Also note that when n is 2 or greater, the just-described triazine compound may have two or more different C(R2R4) moieties. The same rule applies to other similar situations.
Referring to formula (I), a subset of the triazine compounds of this invention is featured by that R1 is NC(RaRb). In these compounds, W can be O; R5 can be H or alkyl; X can be NRc; Rc can be H, methyl, ethyl, or acetyl; Y can be or CH2, and n can be 0, 1, 2, 3, or 4. In some embodiments, R3 is aryl, heteroaryl (e.g., pyridinyl), ORc, SRc, C(O)ORc, or C(O)NRcRd. In other embodiments, R3 is 
in which each of A and Axe2x80x2, independently, is O, S, or NH; each of Re and Rf, independently, is H, alkyl, aryl, or heteroaryl; and m is 1 or 2.
In this subset of triazine compounds, Ra or Rb, preferably, is 
in which B is NRi, O, or S; Bxe2x80x2 is N or CRi; Rg is H, alkyl, or alkoxyl; Rh is halogen, CN, hydroxyl, alkyl, aryl, heteroaryl, alkoxyl, aryloxyl, or heteroaryloxyl; Riis H, alkyl, or alkylcarbonyl; p is 0, 1, or 2; and q is 0, 1, 2, 3, or 4. Preferably, B is NRi; Bxe2x80x2 is CH; Rg is H, methyl, ethyl, methoxy, or ethoxy; Rh is F, Cl, CN, methoxy, methyl, or ethoxy; Ri is H, methyl, ethyl, or acetyl; and q is 0, 1, or 2.
Another subset of the triazine compounds of this invention is featured by that Ri is aryl or heteroaryl. In these compounds, W can be O; R5 can be H or alkyl; X can be NRc; Rccan be H, methyl, ethyl, or acetyl; Y can be O or CH2, and n can be 0, 1, 2, 3, or 4. In some embodiments, R3 is aryl, heteroaryl (e.g., pyridinyl), ORc, SRc, C(O)ORc, or C(O)NRcRd. In other embodiments, R3 is 
in which each of A and Axe2x80x2, independently, is O, S, or NH; each of Re and Rf, independently, is H, alkyl, aryl, or heteroaryl; and m is 1 or 2.
In this second subset of triazine compounds, R1, preferably, is 
in which D is O, S, or NRm; Dxe2x80x2 is N or CRm; Rj is halogen, CN, hydroxyl, alkyl, aryl, heteroaryl, alkoxyl, aryloxyl, or heteroaryloxyl; Rk is aryl or hetereoaryl; R1 is H, alkyl, or alkylcarbonyl; Rm is H, alkyl, or alkylcarbonyl; r is 0, 1, or 2; s is 0 or 1; t is 0, 1, 2, 3, or 4; and u is 0, 1, 2, 3, 4, or 5. Preferably, R1 is 
and Rj is methyl, ethyl, propyl, or benzyl; and r can be 1 or 2.
In another aspect, this invention also features triazine compounds of formula (I), wherein R1 is NC(RaRb), aryl, or heteroaryl; each of R2, R4, and R5, independently, is Rc, halogen, nitro, nitroso, cyano, azide, isothionitro, SRc, or ORc; R3 is Rc, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl, ORc, OC(O)Rc, SO2Rc, S(O)Rc, S(O2)NRcRd, SRc, NRcRd, NRcCORd, NRcC(O)ORd, NRcC(O)NRcRd, NRcSO2Rd, CORc, C(O)ORc, or C(O)NRcRd; n is 0, 1, 2, 3, 4, 5, 6, or 7; X is O, S, S(O), S(O2), or NRc; Y is a covalent bond, CH2, C(O), Cxe2x95x90Nxe2x80x94Rc, Cxe2x95x90Nxe2x80x94ORc, Cxe2x95x90Nxe2x80x94SRc, O, S, S(O), S(O2), or NRc; Z is CH; and W is O, S, S(O), S(O2), NRc, or NC(O)Rc; in which each of Ra and Rb, independently, is H, alkyl, aryl, heteroaryl; and each of Rc and Rd, independently, is H, alkyl, or alkylcarbonyl. A subset of the triazine compounds is featured by that R1 is NC(RaRb); and another subset is featured by that R1 is aryl or heteroaryl.
Alkyl, alkenyl, alkynyl, aryl, heteroaryl (e.g., pyridinyl), cyclyl, heterocyclyl mentioned herein include both substituted and unsubstituted moieties. The term xe2x80x9csubstitutedxe2x80x9d refers to one or more substituents (which may be the same or different), each replacing a hydrogen atom. Examples of substituents include, but are not limited to, halogen, hydroxyl, amino, alkylamino, arylamino, dialkylamino, diarylamino, cyano, nitro, mercapto, carbonyl, carbamido, carbamyl, carboxyl, thioureido, thiocyanato, sulfoamido, C1xcx9cC6 alkyl, C1xcx9cC6 alkenyl, C1xcx9cC6 alkoxy, aryl, heteroaryl, cyclyl, heterocyclyl, wherein alkyl, alkenyl, alkoxy, aryl, heteroaryl cyclyl, and heterocyclyl are optionally substituted with C1xcx9cC6 alkyl, aryl, heteroaryl, halogen, hydroxyl, amino, mercapto, cyano, or nitro. The term xe2x80x9carylxe2x80x9d refers to a hydrocarbon ring system having at least one aromatic ring. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and pyrenyl. The term xe2x80x9cheteroarylxe2x80x9d refers to a hydrocarbon ring system having at least one aromatic ring which contains at least one heteroatom such as O, N, or S. Examples of heteroaryl moieties include, but are not limited to, furyl, fluorenyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridinyl, pyrimidinyl, quinazolinyl, and indolyl.
Set forth below are exemplary compounds (Compounds 1-12) of this invention: 
In still another aspect, this invention features a pharmaceutical composition that contains a pharmaceutically acceptable carrier and an effective amount of at least one of the above-described triazine compounds.
In further another aspect, the present invention features a method for treating an IL-12 overproduction-related disorder (e.g., rheumatoid arthritis, sepsis, Crohn""s disease, multiple Sclerosis, psoriasis, or insulin-dependent diabetes). The method includes administering to a subject in need thereof an effective amount of a triazine compound of formula (I), wherein R1 is NC(RaRb), aryl, or heteroaryl; each of R2, R4, and R5, independently, is Rc, halogen, nitro, nitroso, cyano, azide, isothionitro, SRc, or ORc; R3 is Rc, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl, ORc, OC(O)Rc, SO2Rc, S(O)Rc, S(O2)NRcRd, SRc, NRcRd, NRcCORd, NRcC(O)ORd, NRcC(O)NRcRd, NRcSO2Rd, CORc, C(O)ORc, or C(O)NRcRd; n is 0, 1, 2, 3, 4, 5, 6, or 7; X is O, S, S(O), S(O2), or NRc; Y is a covalent bond, CH2, C(O), Cxe2x95x90Nxe2x80x94Rc, Cxe2x95x90Nxe2x80x94ORc, Cxe2x95x90Nxe2x80x94SRc, O, S, S(O), S(O2), or NRc; Z is N or CH; and W is O, S, S(O), S(O2), NRc, or NC(O)Rc; in which each of Raand Rb, independently, is H, alkyl, aryl, heteroaryl; and each of Rcand Rd, independently, is H, alkyl, or alkylcarbonyl.
The triazine compounds described above include the compounds themselves, as well as their salts and their prodrugs, if applicable. The salts, for example, can be formed between a positively charged substituent (e.g., amino) on a compound and an anion. Suitable anions include, but are not limited to, chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, a negatively charged substituent (e.g., carboxylate) on a compound can form a salt with a cation. Suitable cations include, but are not limited to, sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as teteramethylammonium ion. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing the triazine compounds described above.
In addition, some of the just-described triazine compounds have one or more double bonds, or one or more asymmetric centers. Such compounds can occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z-double bond isomeric forms.
Also within the scope of this invention are a composition containing one or more of the compounds described above for use in treating an IL-12 overproduction-related disorder, and the use of such a composition for the manufacture of a medicament for the just-described use. Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.
The compounds described above can be prepared by methods well known in the art, as well as by the synthetic routes disclosed herein. For example, a triazine compound of this invention (e.g., Compound 1) can be prepared in a stepwise manner by using cyanuric chloride as a starting material and replacing its three chloro groups with various substitutes by the methods described above. Due to the symmetry of cyanuric chloride, the order of displacement is not of particular importance. For example, a chloro group of cyanuric chloride can be substituted with a nucleophile Xxe2x80x94R1xe2x80x94H, wherein X is O or S, thus forming an ether linkage. In another example, a compound of formula (I), wherein Y is CH2 (e.g., Compound 7), can be prepared by reacting the cyanuric chloride with a Grignard reagent, an organotin reagent, an organoboric acid, an organocopper reagent or an organozinc reagent in the presence of an organopalladium compound as a catalyst If preferred, other types of linkages can be prepared by similar nucleophilic reactions. Sensitive moieties on the triazinyl intermediates and on the nucleophiles can be protected prior to coupling. For suitable protecting groups, see, e.g., Greene (1981) Protective Groups in Organic Synthesis, John Wiley and Sons, Inc., New York. A triazine compound thus synthesized can be further purified by flash column chromatography, high performance liquid chromatography, or crystallization.
Also within the scope of this invention is a pharmaceutical composition that contains an effective amount of at least one triazine compound of this invention and a pharmaceutically acceptable carrier. Further, the present invention covers a method of administering an effective amount of one or more triazine compounds described in the xe2x80x9cSummaryxe2x80x9d section to a subject in need of treatment of IL-12 overproduction related diseases (e.g., rheumatoid arthritis, sepsis, or Crohn""s disease, multiple sclerosis, psoriasis, or insulin-dependent diabetes mellitus). xe2x80x9cAn effective amountxe2x80x9d refers to the amount of the compound which is required to confer a therapeutic effect on the treated subject. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described in Freireich et al., (1966) Cancer Chemother Rep 50: 219. Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. An effective amount of the compound of this invention can range from about 0.001 mg/Kg to about 1000 mg/Kg. Effective doses will also vary, as recognized by those skilled in the art, depending on the diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments such as use of other agents.
To practice the method of the present invention, a triazine compound, as a component of a pharmaceutical composition, can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term xe2x80x9cparenteralxe2x80x9d as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
A sterile injectable composition, for example, a sterile injectable aqueous or oleaginous suspension, can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer""s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation. A composition for oral administration can be any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added. A nasal aerosol or inhalation composition can be prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. A triazine compound can also be administered in the form of suppositories for rectal administration.
The carrier in the pharmaceutical composition must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the active ingredient of the formulation (and preferably, capable of stabilizing it) and not deleterious to the subject to be treated. For example, solubilizing agents such as cyclodextrins, which form specific, more soluble complexes with the compounds of this invention, or one or more solubilizing agents, can be utilized as pharmaceutical excipients for delivery of a triazine compound. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and DandC Yellow # 10.
The biological activities of a triazine compound can be evaluated by a number of cell-based assays. One of such assays can be conducted using cells from human peripheral blood cells (PBMC) or human monocytic cell line (THP-1). The cells are stimulated with a combination of human interferon-xcex3 and lipopolysaccharide or a combination of IFNxcex3 and Staphylococcus aureus Cowan I in the presence of a test compound. The level of inhibition of IL-12 production can be measured by determining the amount of p70 by using a sandwich ELISA assay with anti-human IL-12 antibodies. IC50 of the test compound can then be determined. Specifically, PBMC or THP-1 cells are incubated with the test compound. Cell viability was assessed using the bioreduction of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega, Madison, Wis.).
A triazine compound can also be evaluated by animal studies. For example, one of such studies involves the ability of a test compound to treat adjuvant arthritis (i.e., a IL-12 overproduction related disorder) in rats.
Without further elaboration, it is believed that the above description has adequately enabled the present invention. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All of the publications cited herein are hereby incorporated by reference in their entirety.